Fig. 1: L28R is a coding mutation in E. coli DHFR with a unique resistance-conferring mechanism.

a DHFR is an essential enzyme with a central role in the biosynthesis of nucleotides and amino acids. b Trimethoprim (TMP) is a bacteriostatic antibiotic molecule that competitively inhibits DHFR activity. c Resistance-conferring DHFR mutations that reduce TMP affinity are responsible for the evolution of TMP resistance in E. coli. As TMP is a competitive inhibitor of DHFR, resistance-conferring mutations typically reduce both TMP and substrate (DHF) binding affinities. d Frequencies of resistance-conferring DHFR mutations from 40 independent E. coli populations evolved in the morbidostat under TMP selection [7 populations from this study (Fig. 4) and 33 populations from our previous studies^7,16]. L28R is one of the most commonly observed DHFR mutation in laboratory evolution experiments. Different colors on the bar plot are used to represent mutated residues. Stripes with different directionalities are used to distinguish frequency of different amino acid replacements in the same residue. e Unlike other mutations, L28R indirectly increases TMP resistance (higher K_i) by increasing substrate affinity (lower K_m) due to additional interactions with the glutamate tail (green) of DHF (bottom) in comparison with wild-type (top). f X-Ray crystal structure of DHFR^WT (6XG5, resolution: 1.9 Å) bound to TMP drug (blue) and NADPH co-factor (green). g X-Ray crystal structure of DHFR^L28R (6XG4, resolution: 2.1 Å) bound to TMP drug (blue) and NADPH co-factor (green). Omit difference electron density maps for trimethoprim in both structures are provided in Supplementary Fig. 10. h Interaction distances (Å) between DHFR residues (gray) and TMP (blue) for DHFR^WT (black) DHFR^L28R (magenta). i Proposed structure of L28R-specific DHFR inhibitor with modifications (colored spheres) in the hydrophobic tail of TMP while preserving its polar head (red).